Identification of the residues responsible for the alkaline inhibition of Cu, Zn superoxide dismutase: A site‐directed mutagenesis approach

摘要: The catalytic rate of wild type, two single (Lys 120-->Leu, Lys 134-->Thr), and one double 120-->Leu-Lys 134-->Thr) mutants Xenopus laevis B Cu,Zn superoxide dismutase has been studied by pulse radiolysis as a function pH. pH dependence curve the wild-type enzyme can be deconvoluted deprotonation equilibria, at 9.3 (pK1) 11.3 (pK2). Catalytic measurements on indicate that pK1 is mainly due to 120 134, with only minor contribution from other surface basic residues, whereas pK2 titration invariant Arg 141, likely coupled copper-bound water molecule. Accordingly, Brownian dynamics simulations carried out reproduce well rate, when experimentally determined pKs are assigned 120, 141.